title: Can recombinant human glutathione peroxidase 1 with high activity be efficiently produced in Escherichia coli ?
abstract: Cellular glutathione peroxidase 1 (GPx1) is an important antioxidant selenoenzyme. Due to the presence of selenocysteine (Sec) encoded by UGA, normally recognized as a stop codon, the expression of mammalian GPx with traditional recombinant DNA technology is extremely difficult. In this study, a series of human GPx1 (hGPx1) mutants with significantly high catalytic activities were produced for the first time in an Escherichia coli BL21(DE3)cys auxotrophic strain using the single-protein production system. Cys residues in hGPx1 were mutated to Ser in turn because untargeted substitution of Sec in place of Cys resulted in the decline of recombinant selenoenzyme activity. The results of this work showed that the catalytic activities of the mutants increased progressively with decreasing number of noncatalytic Sec residues. Seleno-hGPx1-C2/78/115/156/202S with all Cys residues changed to Ser showed the highest activity (21,268 U/μmol), which was more than 10-fold higher than bovine liver GPx. This increase could be explained by structural analysis of hGPx1 mutants based on homology modeling and binding site analysis. These results lead to the hypothesis that the conversion of noncatalytic Sec residues to Ser may optimize the structure of seleno-GPx in this expression system and consequently increase the catalytic efficiency.
authors.affiliation: 1 College of Pharmaceutical Science, Jilin University , Changchun, China .
authors.forename: Xiao
authors.initials: X
authors.lastname: Guo
authors.forename: Jian
authors.initials: J
authors.lastname: Song
authors.forename: Yang
authors.initials: Y
authors.lastname: Yu
authors.forename: Jingyan
authors.initials: J
authors.lastname: Wei
mesh_terms: D002384:Catalysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005979:Glutathione Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins; D013696:Temperature; D000094765:Glutathione Peroxidase GPX1
chemical_list: D011994:Recombinant Proteins; D005979:Glutathione Peroxidase; D000094765:Glutathione Peroxidase GPX1